Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.
Despite recent treatment advances for chronic hepatitis C virus (HCV) infection, a vaccine is urgently needed for global control of this important liver pathogen. The lack of robust immunocompetent HCV infection models makes it challenging to identify correlates of protection and test vaccine efficacy. However, vigorous CD4+ and CD8+ T-cell responses are detected in patients that spontaneously resolve acute infection, whereas dysfunctional T-cell responses are a hallmark of chronic infection. The HCV p7 protein, forming ion-channels essential for viral assembly and release, has not previously been pursued as a vaccine antigen. Herein, we demonstrated that HCV p7 derived from genotype 1a and 1b sequences are highly immunogenic in mice when employed as overlapping peptides formulated as nanoparticles with the cross-priming adjuvant, CAF09. This approach induced multifunctional cytokine producing CD4+ and CD8+ T-cells targeting regions of p7 that are subject to immune pressure during HCV infection in chimpanzees and humans. Employing a surrogate in vivo challenge model of liver cells co-expressing HCV-p7 and GFP, we found that vaccinated mice cleared transgene expressing cells. This study affirms the potential of a T-cell inducing nanoparticle vaccine platform to target the liver and introduces HCV p7 as a potential target for HCV vaccine explorations.
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